Splenic architecture disruption and parasite-induced splenocyte activation and anergy in Plasmodium falciparum-infected Saimiri sciureus monkeys.

BACKGROUND: The understanding of the mechanisms of immunity in malaria is crucial for the rational development of interventions such as vaccines. During blood stage infection, the spleen is considered to play critical roles in both immunity and immunopathology of Plasmodium falciparum infections. METHODS: Saimiri sciureus monkeys were inoculated with blood stages of P. falciparum (FUP strain) and spleens removed during acute disease (days 7 and 13 of infection) and during convalescence (15 days after start of chloroquine treatment). Cytokine (IFNγ, TNFα, IL2, IL6, IL10, and IL12) responses of splenocytes stimulated with P. falciparum-parasitized red blood cells were assessed by real-time PCR using specific Saimiri primers, and histological changes were evaluated using haematoxylin-eosin and Giemsa-stained slides. RESULTS: Early during infection (day 7, 1-2% parasitaemia), spleens showed disruption of germinal centre architecture with heavy B-cell activation (centroblasts), and splenocytes showed increased expression of IFNγ, IL6 and IL12 upon in vitro stimuli by P. falciparum-parasitized red blood cells (pRBC). Conversely, 15 days after treatment of blood stage infection with chloroquine, splenocytes showed spontaneous in vitro expression of TNFα, IL2, IL6, IL10, and IL12, but not IFNγ, and stimulation with P. falciparum pRBC blocked the expression of all these cytokines. During the acute phase of infection, splenic disarray with disorganized germinal centres was observed. During convalescence, spleens of the chloroquine-treated animals showed white pulp hyperplasia with extensive lymphocyte activation and persistency of heavily haemozoin-laden macrophages throughout the red pulp. CONCLUSIONS: Inability to eliminate haemozoin is likely involved in the persistent lymphocyte activation and in the anergic responses of Saimiri splenocytes to P. falciparum pRBC, with important negative impact in immune responses and implications for the design of malaria vaccine.

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia.

Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

Critical points of Brazil nuts: a beginning for food safety, quality control and Amazon sustainability.

BACKGROUND: One difficulty of self-sustainability is the quality assurance of native products. This research was designed to study the risks and critical control points in the collection, handling and marketing of Brazil nuts from native forests and urban fairs in the Brazilian Amazon by characterisation of morphological aspects of fungi and posterior identification by molecular biology and determination of aflatoxins by high-performance liquid chromatography. RESULTS: Several corrective actions to improve product quality were found to be necessary in both sites. Growth of fungi was observed in 95% of fragments of Brazil nuts from both sites during the between-harvest period. Aflatoxin levels indicated that, although fungal growth was observed in both sites, only Brazil nuts from the native forest showed a high risk to human health (total aflatoxin level of 471.69 µg kg(-1)). CONCLUSION: This study has shown the main issues related to the process design of Brazil nuts, supporting the necessity for research on new strategies to improve the quality of nuts. Also, the habit of eating Brazil nuts stored throughout the year may represent a risk to farmers.

Comparative genetic diversity of wild and captive populations of the bare-faced curassow (Crax fasciolata) based on cross-species microsatellite markers: implications for conservation and management.

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy-Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species' genetic diversity.

Evaluating the diversity of Neotropical anurans using DNA barcodes.

This study tested the effectiveness of COI barcodes for the discrimination of anuran species from the Amazon basin and other Neotropical regions. Barcodes were determined for a total of 59 species, with a further 58 species being included from GenBank. In most cases, distinguishing species using the barcodes was straightforward. Each species had a distinct COI barcode or codes, with intraspecific distances ranging from 0% to 9.9%. However, relatively high intraspecific divergence (11.4-19.4%) was observed in some species, such as Ranitomeya ventrimaculata, Craugastor fitzingeri, Hypsiboas leptolineatus, Scinax fuscomarginatus and Leptodactylus knudseni, which may reflect errors of identification or the presence of a species complex. Intraspecific distances recorded in species for which samples were obtained from GenBank (Engystomops pustulosus, Atelopus varius, Craugastor podiciferus, and Dendropsophus labialis) were greater than those between many pairs of species. Interspecific distances ranged between 11-39%. Overall, the clear differences observed between most intra- and inter-specific distances indicate that the COI barcode is an effective tool for the identification of Neotropical species in most of the cases analyzed in the present study.

Morphological and molecular characterization of Ortleppascaris sp. larvae, parasites of the cane toad Rhinella marina from eastern Amazonia.

This study presents a new record for the occurrence of larval Ortleppascaris sp.(Sprent, 1978). The nematodes were collected from inside fibrous cysts found in the livers of cane toads, Rhinella marina (Linnaeus, 1758), captured in eastern Brazilian Amazonia. This is the first record of Ortleppascaris sp. larvae in both Brazil and this amphibian host, increasing its distribution in South America as well as expanding the number of helminths known to infect this toad. The detailed description of Ortleppascaris sp. provides new taxonomic data for these larvae, as well as sequences of the internal transcribed spacers and small subunit DNA segments, and the cytochrome oxidase I gene, which will, in time, contribute to a better understanding of the phylogeny of this group of parasites.

Proteomics Analysis of the Effects of Cyanate on Chromobacterium violaceum Metabolism.

Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium. The proteome of cells grown with and without cyanate was compared on 2-D gels. Differential spots were digested and identified by mass spectrometry. The bacterium was able to grow at concentrations of up to 1 mM cyanate. Eighteen spots were differentially expressed in the presence of cyanate, of which 16 were downregulated and only two were upregulated. An additional 12 spots were detected only in extracts of cells unexposed to cyanate, and one was expressed only by the exposed cells. Fourteen spots were identified, corresponding to 13 different proteins. We conclude that cyanate promotes expression of enzymes that combat oxidative stress and represses enzymes of the citric acid cycle, strongly affecting the energetic metabolism of the cell. Other proteins that were under-expressed in bacteria exposed to cyanate are involved in amino-acid metabolism or are hypothetical proteins, demonstrating that cyanate also affects expression of genes that are not part of the cyn operon.

Genetic structure of red-handed howler monkey populations in the fragmented landscape of Eastern Brazilian Amazonia.

We genotyped 15 microsatellite loci in order to evaluate the effects of habitat fragmentation, caused by flooding of the Tucuruí reservoir, on the genetic structure of Alouatta belzebul in eastern Amazonia. The analysis included two populations sampled in 1984, representing both margins of the Tocantins river, and three populations sampled 18 years later. Minimal differences in the diversity levels between present-day (Ho = 0.62-0.69 and A(R) = 6.07-7.21) and pre-flooding (Ho = 0.60-0.62 and A (R) = 6.27-6.77) populations indicated there was no significant loss of genetic variability, possibly because of successful management strategies applied during the flooding. The changes observed were limited to shifts in the composition of alleles, which presumably reflect the admixture of subpopulations during flooding. Given this, there were significant differences in the Rst values (p = 0.05) in all but one between-site comparison. Both present-day and original populations showed a deficit of heterozygotes, which suggests that this may be typical of the species, at least at a local level, perhaps because of specific ecological characteristics. The relatively large number of private alleles recorded in all populations may be a consequence of the Wahlund effect resulting from population admixture or a process of expansion rather than the loss of rare alleles through genetic drift. Additionally, the levels of genetic variability observed in this study were higher than those reported for other species of Neotropical primates, suggesting good fitness levels in these A. belzebul populations. Regular genetic monitoring of remnant populations, especially on islands, should nevertheless be an integral component of long-term management strategies.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 May 2009-31 July 2009.

This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediae sp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.

Diagnóstico molecular da infecção por hemoplasmas em gatos domésticos naturalmente infectados da cidade de Belém, Pará / Molecular diagnosis of haemoplasmas infection in naturally infected domestic cats from Belém, Pará

Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' e 'Candidatus Mycoplasma turicensis' são os agentes causadores da micoplasmose felina, que podem causar anemia aguda ou crônica. O objetivo deste trabalho foi determinar a ocorrência de hemoplasmas em gatos domésticos de Belém, Pará. Para isso, 201 gatos foram divididos em três grupos: Grupo A foi composto por 101 gatos de rua capturados pelo Centro de Controle de Zoonoses, o grupo B foi composto por 62 gatos domiciliados e saudáveis e o grupo C foi composto por 38 gatos domiciliados que apresentavam alguma afecção clínica. Foram coletadas amostras de sangue para a realização de Reação em Cadeia pela Polimerase (PCR) para detectar o DNA destes agentes, os quais foram sequenciados e alinhados. A análise estatística foi realizada para detectar a associação entre a infecção, o sexo dos animais e os grupos experimentais. O DNA de pelo menos uma das espécies de hemoplasmas pesquisados foi detectado em 19,9% (40/201) das amostras, sendo o DNA de 'Candidatus M. haemominutum' encontrado em 7,96% (16/201) das amostras, M. haemofelis em 1,49% (3/201) das amostras, enquanto que o DNA de 'Candidatus M. turicensis' foi detectado em 12,93% (26/201) das amostras. O DNA destes três agentes foi detectado em gatos dos grupos A e C, enquanto que no grupo B foi detectado apenas 'Candidatus M. turicensis' e 'Candidatus M. haemominutum' Foi detectada a influência do sexo sobre a infecção hemoplasmas apenas entre 'Candidatus M. haemominutum' e machos. Estes resultados mostraram que os hemoplasmas circulam entre os gatos domésticos em Belém e 'Candidatus M. turicensis' e 'Candidatus M. haemominutum' foram mais comuns do que M. haemofelis, especialmente em gatos vadios.

Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis' are the causative agent of the feline mycoplasmosis, which could cause acute or chronic anemia. The aim of this work was to determine the occurrence of hemoplamas in domestic cats from Belém, Pará. To this, 201 cats were divided into three groups: Group A were composed by 101 stray cats captured by Zoonosis Control Center, group B were composed by 62 owners healthy cats and group C were composed by 38 owners cats that were suffering by some medical condition. Blood samples were collected to perform Polymerase Chain Reaction (PCR) to detect the DNA of these agents, which were sequenced and aligned. Statistical analysis was performed to detect association between the infection, the sex of the animals and experimental groups. The DNA of at least one of the hemoplasmas studied were detected in 19,9% (40/201) of the samples, being the DNA of 'Candidatus M. haemominutum' was found in 7.96% (16/201) of samples, M. haemofelis in 1.49% (3/201) of samples, while 'Candidatus M. turicensis' in 12.93% (26/201) of the samples. The DNA of these three agents was detected in cats from groups A and C, while in Group B was detected only 'Candidatus M. turicensis' and 'Candidatus M. haemominutum'. The influence of sex on hemoplasma infection was detected only between 'Candidatus M. haemominutum' and males. These findings showed that hemoplasma circulate among domestic cats in Belém, and 'Candidatus M. turicensis' and 'Candidatus M. haemominutum' were more common than M. haemofelis, especially in stray cats.

Genetic structure of red-handed howler monkey populations in the fragmented landscape of Eastern Brazilian Amazonia

We genotyped 15 microsatellite loci in order to evaluate the effects of habitat fragmentation, caused by flooding of the Tucuruí reservoir, on the genetic structure of Alouatta belzebul in eastern Amazonia. The analysis included two populations sampled in 1984, representing both margins of the Tocantins river, and three populations sampled 18 years later. Minimal differences in the diversity levels between present-day (Ho = 0.62-0.69 and A R = 6.07-7.21) and pre-flooding (Ho = 0.60-0.62 and A R = 6.27-6.77) populations indicated there was no significant loss of genetic variability, possibly because of successful management strategies applied during the flooding. The changes observed were limited to shifts in the composition of alleles, which presumably reflect the admixture of subpopulations during flooding. Given this, there were significant differences in the Rst values (p = 0.05) in all but one between-site comparison. Both present-day and original populations showed a deficit of heterozygotes, which suggests that this may be typical of the species, at least at a local level, perhaps because of specific ecological characteristics. The relatively large number of private alleles recorded in all populations may be a consequence of the Wahlund effect resulting from population admixture or a process of expansion rather than the loss of rare alleles through genetic drift. Additionally, the levels of genetic variability observed in this study were higher than those reported for other species of Neotropical primates, suggesting good fitness levels in these A. belzebul populations. Regular genetic monitoring of remnant populations, especially on islands, should nevertheless be an integral component of long-term management strategies.

Genetic variability and efficiency of DNA microsatellite markers for paternity testing in horse breeds from the Brazilian Marajó archipelago

In this study, 15 microsatellite DNA loci used in comparative tests by the International Society for Animal Genetics were applied to the evaluation of genetic diversity and management, and the efficiency of paternity testing in Marajoara horses and Puruca ponies from the Marajó Archipelago. Based on the genotyping of 93 animals, mean allelic diversity was estimated as 9.14 and 7.00 for the Marajoara and Puruca breeds, respectively. While these values are similar to those recorded in most European breeds, mean levels of heterozygosity were much lower (Marajoara 49 percent, Puruca 40 percent), probably as a result of high levels of inbreeding in the Marajó populations. The mean informative polymorphic content of this 15-marker system was over 50 percent in both breeds, and was slightly higher in the Marajoara horses. The discriminative power and exclusion probabilities derived from this system were over 99 percent for both populations, emphasizing the efficacy of these markers for paternity testing and genetic management in the two breeds.